The development of mammalian cell culture techniques has revolutionized biological research. Cell culture systems can be used to test new drugs for toxicity or efficacy at an early stage in development when human clinical testing would be high-risk; to produce complex human proteins for therapeutic applications, such as monoclonal antibodies; and as a platform for cell-based therapeutics in the context of adult and embryonic stem cell cultures. In addition, the ability to introduce heterologous recombinant DNA into cultured cell lines gives scientists a powerful adjunct tool for manipulating animal systems for experimentation.
Recently, the need to address regulatory concerns about contamination of cell lines used to express biomolecules and subsequently to manufacture therapeutic products has become more critical. The biotechnology industry as a whole is moving away from the use of FBS-supplemented media for commercial cell culturing in order to ensure that potential animal pathogens or disease-causing animal proteins are not introduced into the human population via future biologics. Serum-free media are becoming the standard protocol for culturing mammalian cells, especially those used for gene expression and protein purification within biologic product pipelines.
The NS-0 mouse myeloma cell line is commonly used in protein expression systems, such as Lonza's GS Gene Expression System (Lonza Group, Basel, Switzerland). The GS Gene Expression System exploits the inability of NS-0 cells to produce enough glutamine to survive without adding exogenous glutamine to the growth medium, by using the enzyme glutamine synthetase (GS) as a marker for cellular transfection with a plasmid vector.
NS-0 cells are also cholesterol auxotrophs, resulting in a situation where media used to support the growth of the cell line must be supplemented with both cholesterol and glutamine. Adding exogenous cholesterol to aqueous media is an intricate and time-consuming process because the lipid must be coupled to sugar moieties, such as cyclodextrins, in order to increase aqueous solubility. In addition, the coupling process is inherently unstable, resulting in cholesterol-supplemented growth media with a very short shelf-life. Further, when chemically defined, serum-free media (CD-SFM) are used instead of fetal bovine serum-supplemented media (FBS) to culture cholesterol-auxotrophic cell lines, cholesterol precipitation occurs frequently. Finally, cholesterol cannot be easily filtered through small pore sterilizing membranes, such as PES, due to its inherent affinity to such polymers, thus significantly reducing the amount of cholesterol in the final filtered medium. This issue further contributes to inconsistencies in batch to batch preparations of cholesterol supplemented media. Addition of exogenous cholesterol directly to medium without passing through a filter also increases the possibility of introducing contaminants, such as adventitious agents and/or endotoxins.
The molecular mechanism underlying the cholesterol auxotrophy of NS-0 cells has recently been identified and characterized (European Collection of Cell Cultures—ECACC, No. 85110503, Deposited by Dr J Jarvis, MRC Laboratory of Molecular Biology, Cambridge, 73B(3) Methods in Enzymology (1981). The cell line does not express the gene coding for an enzyme that catalyzes a step in the endogenous biosynthesis of cholesterol. An enzyme called 3-ketosteroid reductase (3-KSR), one member of a large family of beta-hydroxysteroid dehydrogenases, is encoded by the specific gene. The 3-KSR protein catalyzes the conversion of zymosterone into zymosterol, a precursor of cholesterol. Marijanovic et al., 17(9) MOL. ENDOCRINOL. 1715-25 (2003).
A number of different techniques have been proposed to address the cholesterol auxotrophy of NS-0 cells with the goal of making the culturing protocol more efficient and less dependent on successful cholesterol solubility in aqueous media. One approach has been to use plant-derived or synthetic lipids instead of animal-derived cholesterol to supplement CD media. Gorfien et al., 16(5) BIOTECHNOL. PROG. 682-7 (2000). Another approach has been to engineer NS-0 cells to overexpress 3-KSR, thereby reverting the enzymatic deficiency and allowing the cell line to be cultured without the addition of exogenous cholesterol. Seth et al., 121(2) J. BIOTECHNOL. 241-52 (2006). Finally, researchers are investigating the molecular basis for 3-KSR gene inactivity in NS-0 cells and attempting to restore expression of the gene by demethylating a critical upstream regulatory region, which would relieve the transcriptional repression of the 3-KSR gene. Seth et al., 93(4) BIOTECHNOL. BIOENG. 820-27 (2006).
Nevertheless, there remains a great need in the biotechnology industry for generating stable cell lines through transfection with expression vectors in chemically defined/serum-free media, which are capable of expressing heterologous molecules. Moreover, it is highly desired that the chemically defined/serum-free media is usable off-the-shelf without the need for problematic addition of exogenous cholesterol. The present invention provides an expression vector containing a gene or polynucleotide encoding an enzyme in the sterol or cholesterol biosynthetic pathway of a eukaryotic cell that imparts the ability of the cell to survive and be cultured on chemically defined and/or serum-free media. When this expression vector also comprises a gene or polynucleotide encoding a heterologous protein, polypeptide or peptide, this vector is useful as a metabolic selection marker to select the transfected cells that contain the gene encoding the heterologous protein that can be produced in chemically defined and/or serum-free media.